Method for monitoring nucleic acid assays using synthetic internal controls with reversed nucleotide sequences

ABSTRACT

The present invention relates to methods and compositions that provide a positive control to identify inhibition during a signal amplification reaction. The methods and compositions of the present invention are designed to run in the same tube or assay environment as the experimental or target sample and contain a copy of the target sequence in an inverted form.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/063,922 and U.S. Non-Provisional Application No. 09/183,866,filed Oct. 30, 1998.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to the use of an internal positivecontrol containing an inverse sequence to detect inhibition and toprovide an internal quantitation standard in a nucleic acid assay.

[0003] Modern nucleic acid assay techniques allow researchers andclinicians to detect molecules of interest that are present in extremelylow concentration. These assays use probes to specifically amplify byseveral orders of magnitude and detect the amount of the molecule ofinterest. However, when used diagnostically, falsely negative resultsarising from inhibition of the assay reaction dramatically reduce thepredictive value of the assay. Thus there is a strong need for a methodto control for inhibition of the assay reaction.

[0004] The Polymerase Chain Reaction (PCR) is an example of such anamplification technique for the detection of target molecules. With PCRit is possible to test blood samples for minute quantities of nucleicacid from pathogens, such as the human immunodeficiency virus (HIV). Thetechnique can also be used to detect a variety of different infectiousagents in a number of different clinical settings such as testing bloodor donor organs for infection. Negative results may be unreliable giventhe susceptibility of these techniques to non-specific inhibition by avariety of compounds. Thus, there is a requirement for methods todifferentiate true negative results from false negative resultssecondary to inhibition of the assay.

[0005] For the foregoing reasons, there is a need for an accuratereproducible positive control to detect inhibition in the PCR reaction.The method of detecting inhibition is further applicable to other signalamplification assays.

SUMMARY OF THE INVENTION

[0006] The present invention relates to compositions and methods thatprovide a positive control to identify inhibition during a signalamplification reaction. The methods and compositions of the presentinvention are designed to run in the same tube or assay environment asthe experimental or target sample and contain a copy of the targetsequence in an inverted form.

[0007] One embodiment of the present invention, provides for an internalcontrol cassette for use in a polynucleotide detection assay in which atarget sequence is detected. The target sequence has primer bindingsites flanking an internal target sequence. The cassettes comprisesprimer binding sites flanking an internal control sequence, wherein theinternal control sequence comprises said internal target sequence in areversed orientation.

[0008] In one aspect of this embodiment, the internal control cassettefurther comprises one or more primer binding sites adjacent to theinternal control sequence. In another aspect, the internal controlcassette further comprises the nucleic acid sequence of SEQ. ID. NO. 5.The internal control cassette may be a component of a plasmid.

[0009] Another embodiment of the present invention contemplates a methodfor detecting signal amplification inhibition in an assay comprising thesteps of: co-amplifying a target sequence and an internal controlcassette, wherein the internal control cassette comprises the targetsequence in a reverse orientation. Assays contemplated for use with thepresent invention are selected from the group consisting of PCR,real-time PCR, branched DNA (bDNA)-based signal amplification assays,nucleic acid sequence based amplification assays (NASBA), andtranscription mediated amplification (TMA).

[0010] The target sequences usable in the present invention include anynucleic acid sequence that may be assayed with techniques known in theart. In one aspect of this embodiment, the target sequence comprises DNAor RNA. In another aspect, the target sequence is a nucleic acidsequence from a virus selected from the group consisting of HSV, HIV,HCV, CMV, and HPV. In another aspect, the target sequence comprises thenucleic acid sequence of SEQ. ID. NO. 1.

[0011] Similarly, the internal control cassette sequences include anysequence that may be a target sequence. For example, an internal controlcassette comprises the nucleic acid sequence of SEQ. ID. NO. 2.

[0012] The methods of the present invention further comprise the step ofassaying products generated by the co-amplification described above. Thepresent invention further contemplates an additional step of assayingproducts by a primer binding assay comprising the step of determiningthe extent of product binding to a capture probe specific for theinternal control cassette product. In one aspect of this embodiment, thecapture probe consists of the nucleic acid of SEQ ID NO. 6.

[0013] Another embodiment of the present invention contemplates a methodfor detecting signal amplification inhibition in an assay comprising thesteps of contacting one or more hybridization probes with both a targetsequence and an internal control cassette in the same medium, whereinthe internal control cassette comprises the target sequence in a reverseorientation. For example, the assay of this embodiment is a molecularbeacon assay. In another aspect, the internal control cassette comprisesthe nucleic acid sequence of SEQ. ID. NO. 2. In still another aspect,the hybridization probe comprises the nucleic acid sequence of SEQ. ID.NO. 8.

DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 graphically depicts the HSV gB target and ICC sequences.

[0015]FIG. 2 graphically depicts the amount of product produced in a PCRreaction. The shaded bars represent HSV-related product and the filledbars represent ICC-related product. The magnitude of the bar are theoptical density of the samples read at 450 nm.

[0016]FIG. 3 graphically depicts the amount of product produced in a PCRreaction and the specificity of the signals produced therein. The shadedbars represent HSV-related product and the filled bars representICC-related product. The magnitude of the bar are the optical density ofthe samples read at 450 nm.

[0017]FIG. 4 graphically depicts a comparison of samples assayed usingthe Flowmetrix assay of Example 5 against the microtiter based detectionsystem of Example 3.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention relates to methods and compositions thatprovide a positive control to identify inhibition during a signalamplification reaction. The methods and compositions of the presentinvention are designed to run in the same tube or assay environment asthe experimental or target sample and contain a copy of the targetsequence in an inverted form.

[0019] The methods and compositions of the present invention offers anumber of advantages over other methods of controlling the variables ofsignal amplification assays. One benefit of the present invention'sdesign relates to the possible presence of inhibitors in the reactionmix. The methods described herein provide the means to control forinhibition in the experimental reaction.

[0020] Traditionally, a signal amplification assay is conducted with anexperimental sample and positive and negative controls run in separatetubes, wells, or assay environments. The goal of these assays is toexamine the experimental sample for the presence or absence of a targetsequence. The positive control sample provides a check to insure thefunctionality of the assay's reagents. The negative control sampleprovides a means to determine the background signal.

[0021] After the assay is run and the results are generated, one ofskill in the art will examine the experimental well for a signal. Thepresence or absence of a signal indicates the presence or absence of thetarget molecule. In the positive control sample, the presence or absenceof a signal indicates whether or not the assay's reagents arefunctional. Thus, if there is no signal in the experimental well but asignal in the control well, one of skill in the art may conclude thatthere is no target sequence in the experimental well, since the resultfrom the control well indicates that the assay's reagents arefunctional.

[0022] This conclusion will be accurate if the reason for the absence ofsignal from the experimental well is that there is no target sequencecontained therein. If, on the other hand, there are contaminants in theexperimental sample that result in the inhibition of the signalamplification reaction, then no signal will be produced even though atarget sequence is present in the experiment sample. The positivecontrol sample will produce a signal, indicating that the reagents usedin the assay are functional. Under these conditions, one of ordinaryskill in the art could incorrectly interpret the results from signalamplification assay as a negative result.

[0023] The methods and compositions of the present invention aresuperior to traditional standards because, by combining experimental andcontrol fragments into one environment, one is capable of detecting thepresence of an inhibitor in the reaction mixture. This capability isespecially important in the clinical setting. In a conventional priorart positive control system, when a blood sample is tested for thepresence of a particular target molecule, such as a viral nucleic acids,the experimental and control reactions are tested separately. A positivecontrol sample is run along side the experimental sample to insure thatthe reagents used in the reaction are functioning properly. For example,the experimental tube could contain a sample of blood, the amplificationprimers, and the other reagents. In a separate tube a positive controlreaction is set up using a known target sequence, appropriate primers,and a sample of the same reagents used in the experimental tube. Afterthe PCR reaction is complete, the results are examined.

[0024] If a positive control for inhibition is included in theexperimental tube, (i.e., an internal positive control) then theclinician can more confidently determine whether the target molecule wasactually present, or whether the results obtained were merely a falsenegative. If no signal is obtained from the assay's experimental well,then there was inhibition of the assay reaction. If a signal is obtainedfrom only the internal positive control but not from the targetmolecule, then it is reasonable to conclude that there was no targetmolecule present in the sample. A benefit of the present invention isthat it permits an investigator to differentiate a true negative resultfrom a false negative caused by inhibition in the experimental reactionsample.

[0025] The present invention contemplates utility for use as an internalinhibition control in a variety of signal amplification assays. Examplesof signal amplification assays include: the polymerase chain reaction(PCR), variations of PCR, including reverse transcriptase PCR, real-timePCR, branched DNA (bDNA) assays, nucleic acid sequence basedamplification assays (NASBA), transcription mediated amplification(TMA), cytoflowmetric assays, molecular beacon assays, hydridizationreactions, and detection assays.

[0026] The internal control cassette (ICC) may consist of any targetsequence that may be amplified by PCR or other nucleotide amplificationtechniques. Sequences that are present in clinically important diseasestates are particularly relevant to the present invention. Examplesprovided for illustrative purposes include sequences from viruses suchas human immunodeficiency virus (HIV), herpes simplex virus (HSV),hepatitis C virus (HCV), human papilloma virus (HPV), andcytomegalovirus (CMV). Examples of particular genes that may be used astarget sequences include the HSV gB gene and the HIV gp24 and gag genes.

[0027] As discussed above, the present invention provides a method tocontrol for inhibition of signal development in an experimental wellwithin a signal amplification assay. The ICC of the present inventionprovides the template from which the internal control signal isgenerated.

[0028] The ICC constructs of the present invention may be comprised of anumber of elements used to generate the control signal. The ICCadvantageously comprises a target sequence with an internal segment inan inverted orientation, with respect to the target sequence as itappears in nature, and one or more primer binding sites. The segment ofsequence inverted in the ICC preferably lies immediately adjacent to oris flanked by the amplification primer binding site or sites that areused to amplify the experimental target sequence.

[0029] The entire construct may be a plasmid or some other replicatingconstruct. Altenrantivly, the ICC may be a linear fragment of nucleicacid or polymerized amino acids. When the ICC is a plasmid, the plasmidmay be of bacterial origins.

[0030] A plasmid used to construct the ICC can advantageously containall of the necessay components found in any bacterial plasmid used inthe field of molecular biology. For example, an origin of replicationwhich would permit it to replicate within host bacteria may be included.Also, a suitable plasmid may contain a drug resistance marker, such asthe ampicillin or tetracycline resistant markers. Further, a suitableplasmid may contain a multiple cloning site to facilitate the cloning inof the target sequence to be used in the ICC. Other necessary or usefulplasmid components may also be included in a plasmid used to constructan ICC, according to the judgment exercised by one of ordinary skilledin the art.

[0031] The present invention also contemplates the use of an internalcontrol for RNA signal amplification assays. In a preferred embodiment,a control plasmid for use in an RNA signal amplification assay isprepared as described above. In another preferred embodiment, aninducible promoter (such as lac) and a sequence such as a poly(A) tailare cloned at the 5′ and 3′ ends of the inverted target sequence(respectively) in the multiple cloning site of the internal controlplasmid. Using this plasmid it is possible to produce RNA molecules (viain vitro transcription) which contain the inverted capture sequence of aparticular target gene and as such would provide a suitable reagent withwhich to control for inhibition of a RNA based amplification reaction.

[0032] Other methods of controlling for inhibition often use randomsequences of nucleotides as the target for the control reaction.Nevertheless, use of random sequences does not accurately reflect thebiochemical limitations that come into play during the amplification ofa particular target sequence. These differences, for example, indifferences in nucleotide frequency or differences in the overallchemical nature between a target sequence and a control sequence mayhave a significant impact of the final yield.

[0033] The present method differs from those methods of the prior art inthat it uses the same sequence as the target in the internal controlconstruct, although an internal segment is inverted. By using aninverted sequence of a proposed target, as opposed to a randomlygenerated control sequence, the present invention creates a controlsequence that shares many of the same biochemical characteristics of thetarget sequence (e.g., reaction kinetics, temperature of melting(T_(M)), and nucleotide composition).

[0034] One shared feature is that the same primers may be used toamplify both the control and target sequences. When the same primer pairis used for both sequences, the hybridization or primer annealingconditions for both the experimental and ICC control sequences are thesame. Thus, using the same primers and primer binding sites for bothsequences eliminates another variable which might effect the signalproduced from the control and target sequences.

[0035] The choice of primers, their length and coding sequence are apreference of one of ordinary skill in the art. For example, when theprimers are for use in a PCR reaction, the primers may be about 5 to 50nucleotides long. Alternatively, each primer may be about 10 to 40nucleotides long. In yet another alternative, each primer may be 15 to35 nucleotides in length.

[0036] Using the inverted control sequence of the present invention alsoprovides a number of quantitative similarities between the sequencesthat improves the significance of the inhibition control. Thisquantitative sequence similarity between target and control ICCsequences provides a number of advantages over conventional methods. Forexample, since the amplified sequences of the target and controlplasmids are of the same length and composed of the same nucleotidebases, the reaction parameters for the two plasmids are identical.Reaction parameters such as the T_(M), the length of the sequenceamplified, primer annealing or hybridization and primer usage are allsubstantially the same for the experimental and control sequences of thepresent invention. Given the similarity in reaction parameters betweenthe two sequences, the yield of the co-amplification reactions shouldalso be similar. Thus the inverted sequence of the control plasmidprovides an extremely valid method for investigators to monitor forinhibition during signal amplification reactions.

[0037] The present invention allows an investigator to control forinhibition of a sequence amplification reaction with a control sequencethat has the same T_(M) as the target sample. The T_(M) of interactingnucleic acid strands is determined by their sequence. Here, the controland target sequences both have the same composition of nucleotide basepairs arranged in a the same sequence, albeit inverted. Therefore, theinternal control and target sequences have the same T_(M).

[0038] The importance of using a control sequence that binds assayprimers with the same T_(M) as the target sample becomes clear when thesteps of the assay method are examined. For example, during PCRsubsequent rounds of denaturation, annealing and polymerization are usedto create a PCR product. During the denaturation step, as thetemperature of the rises, the forces which hold the target strandstogether will be insufficient to keep the molecule double stranded. Whenthe template becomes single stranded, it is available to bind theprimers and the enzyme of the PCR reaction.

[0039] Sequences that have higher T_(M)s may require more heat to serveas efficient templates in the PCR reaction. This comes from the factthat G, C, A, and T, each bind to each other with either 2 or 3 hydrogenbonds. Thus, one utilizes a less accurate control when the positivecontrol sequence and its corresponding primer or probe has a differentTm than that of the experimental sample and its corresponding primer orprobe. Accordingly, one should avoid merely random sequences.

[0040] Another important feature of the present invention is the lengthand composition of the control sequence. The length and composition ofthe sequence amplified may effect the signal obtained from the assay.During polymerization, the action of the synthesizing enzyme travelingdown the template strand is known as processivity. The processivity ofthe enzyme may decrease as the length of the target sequence increases.So, the longer the target sequences, the more likely that it is that thePCR enzyme will fall off the template before completing the synthesis ofthe replicated strand. Premature termination of polymerization resultsin a product that differs in length from the target sequence and thatdifference could be misread as a negative result.

[0041] The positive control of the present invention eliminates thisproblem. Since the amplified region of the positive control plasmid isthe same length as the target sequence, the rate of prematuretermination should be the same for both sequences. As a result, if thereare apparent qualities of the target sequence which cause the PCR enzymeto fall off, those same characteristics should be present in theinverted control sequence and the PCR enzyme should fall off thatsequence as well.

[0042] Particular embodiments of the invention are discussed in detailbelow. The following examples are for illustrative purposes only andshould not be interpreted as limitations of the claimed invention. Thereare a variety of alternative techniques and procedures available tothose of skill in the art that would similarly permit one tosuccessfully perform the intended invention.

EXAMPLE 1

[0043] Internal Inhibition Control Plasmid Construction

[0044] Construction of an internal control cassette (ICC) involvedcreating a DNA fragment containing a portion of the HSV (herpes simplexvirus) gB gene (148 base pairs, nucleotide numbers 797-945), with thecentral 39 base pairs (nucleotide numbers 859-898) in the reverseorientation. (See FIG. 1). The gB gene was discussed in Stuve, et al.,“Structure and expression of the herpes simplex virus type 2glycoprotein gB gene.” J. Virol., 61(2):326-335 (1987) and Bzik, et al.,“Nucleotide sequence specifying the glycoprotein gene, gB of herpessimplex virus type 1.” Virology 133:301-314 (1984)(herein incorporatedby reference). See also, Sutton, et al., Transgenic Research 1:228-236.(1992). The total length of the ICC fragment was 160 base pairs whichincludes the 148 base pair region and a unique restriction endonucleasesite on each terminus of the fragment (5′=EcoRI, 3′=Xhol) for cloningpurposes.

[0045] De novo construction of the HSV-ICC fragment was performed usingPAGE purified oligodeoxynucleotides, a ligase chain reaction developedfor synthetic gene construction (modified from Sutton, et al., 1992) anda thermostable ligase (Ampligase™, Epicentre Technologies, Madison,Wis.). After construction and cloning, sequence verification wasperformed by dye-terminator sequence chemistry (ABI 337).

[0046] A claiming plasmid pBluescript KS (Stratagene, San Diego, Calif.)was chosen to carry the internal control sequence. Any plasmid capableof replication in bacteria and suitable for molecular biologicalmanipulation may serve. The multiple cloning region of the controlplasmid was then cut with restriction endonucleases which correspond tothe sites on the de novo constructed DNA fragment (which contained thecentral reversed capture region). The restriction enzyme digestioncreated a linear plasmid that accepted the exogenous nucleic acid samplethat was the internal control sequence. The digested control plasmid wasthen isolated from the digestion reaction using standard techniquesknown to those with ordinary skill in the art. Those techniques includebut are not limited to the use of glass beads, various column matrices,gel isolation, etc.

[0047] The purified cleaved plasmid was then mixed with the purifiedfragment and the DNA molecules were ligated together using standardtechniques known in the art. A DNA ligase was used to close thephosphate backbone of the newly formed internal control plasmid.

EXAMPLE 2

[0048] Internal Inhibition Control for the Polymerase Chain Reaction(PCR)

[0049] Example 2 below discusses the inhibition detection technology ofthe present invention for use with the polymerase chain reaction. Thismethod recites the use of a target nucleotide sequence and a pair ofprimers that are complementary to that sequence. The method discussedbelow also comprises the use of an internal control plasmid in which thecontrol sequence is the inverted nucleotide sequence of the targetsequence. The primers used in the reaction are complementary to both thetarget sequence and to the internal control construct. PCR was thenperformed on the mixture.

[0050] In the PCR reaction, a target sequence is amplified by severalorders of magnitude using a template, a pair of primers and a DNApolymerase enzyme. Typically, a control reaction is run side-by-sidewith the experimental reaction to determine the functionality of thereagents. PCR provides a powerful tool for detecting small quantities ofa target nucleic acid sequence in solution. Nevertheless, the use of atraditional positive control does not provide an investigatorinformation as to whether or not the polymerization reactions in theexperimental tube has been inhibited. The protocol described belowincludes the internal inhibition control of the present invention thatpermits the investigator to detect the presence of signal amplificationinhibition.

[0051] Once the templates, primers and reaction reagents are collectedand mixed, sufficient heat is applied to the sample to denature thedouble stranded complex. Polymerization primers are then permitted toanneal to the denatured template. The primer-template complex of the PCRreaction is then bound by the DNA polymerase enzyme. The Taq polymeraseis an example of a PCR suitable DNA polymerase. The DNA polymerase thenproceeds to synthesize a polynucleotide that is complementary to thetarget strand. After a given period of time the enzyme is disassociatedfrom the template. The cycle is then repeated. Repetition of this methodpermits the amplification of a given sequence as many as 4×10⁶ times intwenty-five cycles of the PCR reaction. The PCR reaction is described infurther detail in Mullis (1987) U.S. Pat. No. 4,683,202, hereinincorporated by reference.

[0052] A polymerase chain reaction is performed using the protocoldescribed below. This protocol is based on from Current Protocols inMolecule Biology Volume 2, Chapter 15.1 (1995), which is incorporatedherein by reference. A PCR amplification buffer concentrated 10 fold isprepared. The 10×PCR amplification buffer contains: 500 mM KCl, 100 mMTris-HCl, pH 9.0 (at 25° C.); 0.1% Triton X-100. The four nucleotidetriphosphates (dNTPs) are mixed for ease of application. For example,the 2.5 mM 4dNTP mix is made by combining equal volumes of each dNTPs ata concentration of 10 mM. These reagents are all commercially available.Two primers, one in the forward and one in the reverse orientation aredesigned based on standard principles known in the art. These primersare diluted to a concentration of 20-50 pmol/μl in H₂O.

[0053] Template DNA is selected using standard parameters well known inthe art. In this Example, the template consisted of 1 pg of viralgenomic DNA/10 μl. An inhibition control ICC is used in combination withthe 1 pg of genomic DNA a mixture containing approximately 500 copies,bringing the final volume to 10 μl.

[0054] An enzyme suitable for PCR, such as Taq DNA polymerase, is usedat a starting concentration of 5 U/μl. Magnesium chloride has been shownto effect PCR reactions, so three concentrations of the salt wereprepared: (L)15 nM, (M)30 mM and (H)45 mM MgCl₂. Sterile mineral oil wasused to seal the reaction.

[0055] The PCR reactions are performed with the following volumes ofreagents (final concentrations): 10 μl of the 10×PCR amplificationbuffer, 1 μl Primer 1 (0.5 :M), 1 μl Primer 2 (0.5 :M), 10 μl ofTemplate DNA, 2 μl of 10 mM 4 dNTP mix, 0.5 μl of Taq polymerase (2.5U), and H₂O to 90 μl. The protocol requires that 90 μl each of themaster mix be placed into three 0.2 ml tubes labeled L, M, and H. Toeach tube was added 10 μl of each corresponding concentration of MgCl₂so that the final concentrations are 1.5, 3.0 and 4.5 mM, respectively.

[0056] A commercially available thermocycler is used to perform the PCRreaction cycling of temperatures. The following steps listed compose onePCR cycle. The reaction tubes are denatured for 1 minute at 94° C. Next,the primers are annealed to the template between 55 and 60° C. dependingon the T_(M) of the primers.

[0057] The primers are extended on the template at 72° C. for 1 to 3minutes depending on the length of the target sequence. The longer thesequence to be amplified, the longer the extension time. Once the cycleis complete, the thermocycler returns to the denature step. The reactioncycled for 25 to 30 times.

[0058] The results of the PCR reaction are assessed after the reactionis complete. If the positive control shows PCR product by detectionmethods well known in the art, the investigator will know that thereagents used in the reaction are functional. If an experimental signalis present, then the investigator knows that the sample contains themolecule of interest. If, however, there is no experimental signal, thenthe investigator needs to evaluate the signal from the ICC. If a signalis present from the ICC but not from the experimental sample, then theresults are negative and the investigator may conclude that there is nodetectable target sequence in the experimental sample. Alternatively, ifthere is no signal from either the experimental sample or the ICC, thenthe investigator may conclude that some inhibitory factor is present inthe experimental sample and that the negative results may or may notindicate the presence of the target sequence in the experimental sample.

EXAMPLE 3

[0059] Detection of Herpes Simplex Virus Type 1 (HSV-1) and Type 2(HSV-2) in Cerebral Spinal Fluid by Qualitative Polymerase ChainReaction

[0060] This Example details procedures for using a PCR-based assay fordetecting HSV-1 and HSV-2 DNA in cerebral spinal fluid (CSF). Detectionof these viruses is an essential step in determining whether patientsare chronically infected with these specific viruses.

[0061] The CSF used in this assay was obtained by spinal tap accordingto techniques well known in the art. Upon collection of the CSF, thesample was refrigerated at 1-4° C. or frozen at −20° C. if storagelasted more than one week.

[0062] Samples obtained for amplification included CSF from two patientsPatient #21002 and Patient #28350, normal CSF (negative control) andnormal CSF spiked with cultured HSV as a positive control. These sampleswere prepared and co-amplified with an ICC to assay for the presence ofHSV.

[0063] As described in the Example above, a master mix of reagents wasassembled for use in the PCR assay.

[0064] A 250 μl sample of CSF was placed into a 2 ml microcentrifugetube and subjected to centrifugation at 1350 revolutions per minute(RPM) (1730 g) for 5 minutes. Two 100 μl samples of CSF were removedfrom the tube and pipetted into two separate 1.5 ml conicalmicrocentrifuge tubes. The next step was to add 500 μl phosphatebuffered saline (PBS) into all experimental and control tubes. Thissolution was vortexed well to mix the CSF and PBS.

[0065] Control samples were prepared in 1.5 ml conical microcentrifugetubes to monitor the PCR reaction. The positive control consisted of 100μl of 1 μl stock HSV in 10 ml of deionized water (ddH₂O). The negativecontrol consisted of 100 μl of ddH₂O. The marked patient samples andcontrol tubes were centrifuged for 1 hour at 21,000 RPM (39,444 g) at 4°C. At this point there were two samples per patient and and two negativeand positive controls.

[0066] After removing the supernatant from the spun samples, beingcareful not to aspirate any precipitated matter. Approximately 10 μl ofsupernatant remained in the tube after aspiration. Next 10 μl of 0.025%BSA was added to the Lysis Buffer, which contained 0.4% (weight tovolume) tergitol type NP-40; 1.25 mM DTT; and 4,000 copies of ICC/ml. Inturn, 100 μl of this lysing reagent was added to each tube. The tubeswere left to incubate at room temperature for 10 minutes. After theincubation, 50 μl of the solution from each sample was placed into aseparate PCR tube.

[0067] To that tube was added 50 μl of the master mix. The master mixcontained the following reagents: PCR reaction buffer, 25 nM MgCl₂, 10mM dNTP, 20 mM dUTP, 100 μM PCR Primer gB₁ (SEQ ID NO. 3), 100 μM PCRPrimer gB₂ (SEQ ID NO. 4), 1 Unit/μl of HK-UNG (Thermolabile UracilN-Glycosylase; Epicentre Technologies, Madison, Wis.), 5 Units/μl Taq,and ddH₂O. The primers were biotinylated to facilitate product detectionfollowing the amplification reaction. After the addition of the mastermix, the tubes were placed into a thermocycler and amplified. Table 1below describes the steps of the program. TABLE 1 Thermocycler Program #CYCLES TIME AND TEMPERATURE 1 30 minutes at 37° C. 1  3 minute at 95° C.5 95° C. for 45 seconds, 64° C. for 45 seconds, 72° C. for 45 seconds 3095° C. for 15 seconds, 64° C. for 15 seconds, 72° C. for 15 seconds

[0068] After the completion of the amplification, the samples wereremoved from the thermocycler. Denature Solution consisting of 1.6%NaOH, 1 mM EDTA and amaranth dye (Roche)(25 μl) was added to each samplefollowed by mixing. These samples were analyzed for the presence ofreplicated materials and are discussed below.

EXAMPLE 3

[0069] Assay for Determining the Presence of Replication Products

[0070] A microtiter plate based assay was used to determine the presenceor absense of amplification products. This assay utilized a DNA captureprobe to bind to assay the products of the PCR reaction described inExample 2. This microtiter assay may also be used to assay amplificationproducts produced by amplification reactions other than PCR.

[0071] Capture probes, such as the HSV gB probe (SEQ. ID. NO. 5) or theICC probe (SEQ. ID. NO. 6) were coupled to an amino group at the 5′ endsof the capture probes via a six carbon linker. The probes weresynthesized with the linker already attached using standardphosphoamidite chemistry, which is well known in the art. The HSV gBprobe was am antisense probe with a sequence that corresponded tonucleotide positions 1803 to 1841 of the gB gene of both HSV-1 andHSV-2. The ICC capture probe was specific for the ICC PCR product. (SeeFIG. 1).

[0072] The labeled capture probes were applied to the wells of highbinding flat bottom 1×8 strip well microtiter plates. (Corning Costar).To the plate was added 100 μl/well of amine modified capture probe inprobe binding buffer (50 mM Na2PO4, pH 8.5, 1 mM EDTA) at aconcentration of 25 pmol/well or greater. The plate was then incubatedovernight at 4° C. The unbound probe was removed from the plate bywashing the plate three times with PBS. Next, the plate was blocked byadding 200 μl of 3% BSA in the probe binding buffer. This was incubatedfor 30 minutes at 37° C. at which time the solution was decanted.

[0073] The amplified specimen samples, 50 μl per reaction tube, were thetransferred into each well of the prepared plate and mixed 5 times. Theplate incubated for 1 hour at 37° C. Following the incubation the platewas washed five times using 1× wash solution with an automated traywasher. The 1× wash solution consisted of 10 mM phoshate buffer (pH7.2), 150 mM NaCl, 1 mM EDTA, 0.5% PROCLIN 300. To the washed plates wasadded 100 μl/well of conjugate solution. The conjugate solutionconsisted of: 25 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1.25 μg/mlStreptavidin-horseradish peroxidase, and 0.1% (v/v) Tween-20. The platewas incubated for 15 minutes at 37° C. following addition of theconjugate solution. Following this incubation, the plate was againwashed 5 times with 1× wash solution using an automated plate washer. Tothe washed plates was added 100 μl of substrate solution that consistedof 51.4 mM Na₂HPO4, 24.3 mM Citric Acid, 1 mg/ml3,3′,5,5′-Tetramethylbenzidine Dihydrochloride, and 40% (v/v)N.N-Dimethylformamide. Following addition of the substrate, the platewas covered to exclude light, and was incubated at room temperature for5 minutes. After the incubation period, 100 μl of stop solution wasadded to each well. The plate was then read at an optical density of 450nm and the results are shown in FIG. 2.

[0074]FIG. 2 shows the enzymatic activity detected from the samplesprepared in Example 2. Patient #21002 is clearly positive for HSV whilePatient#28350 is negative for HSV viral DNA. Additionally, the controlsindicate that there were no inhibitory substances in the PCR reactionmix, so that the absence of an HSV signal may be confidently interpretedas the lack of HSV target sequence, rather than a false negative. Thisconclusion was supported by the strong signal produced by the ICCsequence in all of the samples tested.

EXAMPLE 4

[0075] Specificity of HSV and ICC Capture Probes

[0076] To address the possibility of cross reactivity between the HSVand ICC capture probes, genomic HSV DNA or purified ICC plasmid DNA wasamplified along with a water based negative control using thebiotinylated primers (SEQ. ID. NOS. 3 and 4) and reaction conditionsdiscussed above in Example 2. At the conclusion of the PCR reaction, thesamples containing the HSV amplicon (SEQ. ID. NO. 1) or the ICC amplicon(SEQ. ID. NO. 2) or no amplicon (negative control) were individuallyalkali denatured (denaturing reagent) and aliquoted into microtiterplate wells and allowed to hybridize in neutralizing buffer. The assaywas performed as described in Example 3.

[0077] The wells contained a solid-phase bound oligonucleotide sequencespecific probe specific for either HSV (SEQ. ID. NO. 5) or the ICCamplicon (SEQ. ID. NO. 6). After the hydridization and washing steps ofthe assay protocol, an avidin-horseradish peroxidase (AV-HRP) reagentwas added to the wells. The AV-HRP bound to the biotin-labeled PCRproducts that were in turn bound to the plate via their interaction withthe capture probes. The bound AV-HRP conjugate present in each well wasdetected by a reaction with peroxide and tetramethylbenzidine to form acolored product. The optical density (OD₄₅₀) was determinedspectrophotometrically. The results of this experiment are shown in FIG.3.

[0078] The data in FIG. 3 show that the HSV PCR product boundspecifically to the HSV capture probe containing wells, while the ICCproduct only bound to the ICC capture probe containing wells. Theseresults show the specificity of the various capture probes for theirtarget sequences.

[0079] Alternative Probe Detection Assays

[0080] One aspect of the present invention contemplates a probe specificfor the internal inhibition control plasmid bound to a fluorescentlylabeled microsphere. For example, probes specific for target sequenceand ICC PCR products, respectively, are used to assay for the presenceof herpes simplex virus (HSV) DNA in a sample using the FlowMetrix™cytometric microsphere technology (Luminex Corp., DeSoto, Tex.).

EXAMPLE 5

[0081] Detection of PCR Products Using Flow Cytometric MicrosphereTechnology

[0082] In this Example, two sequence specific oligonucleotide probes,one for the HSV gB gene PCR product (SEQ ID NO 5) and one for the ICCproduct (SEQ ID NO 6) were individually bound to two different subsetsof microspheres. The microsphere subsets were distinguishable based onunique levels of incorporated orange and red fluorescent dyes. Thesemicrosphere subsets were then mixed to form a multiplex set. Each of thecomplementary probe target pairs represented the internal sequencespecific region within the HSV gB gene and the ICC control plasmid.

[0083] Upon completion of the PCR reaction described in Example 2, theamplification products are hybridized in a multiplex reaction containingboth of the labeled probes and the target microspheres. Fluorescencefrom the probes is produced and detected by a flow cytometer. (Smith etal., Clinical Chemistry 44:2054-2056 (1998); van Huisden, et al., J.Histochem. 45:315-319 (1997)). If there are inhibitory contaminantswithin the PCR reaction mixture, then no signal will be seen from eitherthe target sequence or the control plasmid.

[0084] The presence of the control plasmid allows the investigator todifferentiate a true negative result due to the absence of the targetsequence from a negative result due to endogenous inhibition of the PCRreaction. This Example illustrates the utility of this invention, forwithout the presence of an internal control for inhibition, aninvestigator would be unable to discern whether the negative resultindicated a lack of target or merely internal amplification inhibition.

[0085] To illustrate this aspect of the present invention, a serialdilution series of HSV and ICC PCR products, (in equimolar ratios) wasaliquoted. Each mixed dilution was detected by both the microtitercapture plate assay described in Example 3 and by FlowMetix flowcytometry. The data are shown in FIG. 4. Duplicate samples were analyzedand averaged. Optical densities for the microtiter plate data are on thex-axis while data from FlowMetrix is on the y-axis. The ICC amplicondata is shown in FIG. 4A. HSV amplicon detection data are in FIG. 4B.These results show a high degree of correlation between the twodetection methods.

[0086] Use of the ICC for Amplification Assay Calibration andQuantification

[0087] In another embodiment of the present invention, the ICC may beused as a means to calibrate or quantify the product of an amplificationreaction (signal or target). For example, the addition of a knownquantity of the ICC to an amplification reaction would permit aninvestigator to quantitate the amount of target sequence producedagainst the amount of ICC product produced. The comparison would beespecially meaningful since both products are produced within theexperimental sample tube of the amplification reaction.

[0088] Quantification of a PCR Target Product by Comparison with an ICCStandard

[0089] This Example provides a method to quantify the amount of targetPCR sequence produced during an amplification assay. The comparison ismade possible by knowing the starting concentration of ICC andcalculation of the PCR product over a known amplification cycle profile.The ICC is constructed as discussed above. A number of experimentalreactions may be amplified containing a number of different ICC startingconcentrations. The ICC products may be used as a standard curve withwhich to predict the amount of starting target sequence. As in theprevious examples, the ICC and target templates are present in the sameexperimental tube. The ICC template and primers may also be run in aseparate reaction tube to compare ICC product formation in the presenceand absence of target sample.

[0090] The PCR reaction is run as described in Examples 1 and 2. Theproducts of that reaction are analyzed as described in Example 3. ICCproducts produced in the experimental and control tubes are compared andquantified. The amount of product produced by the ICC amplifications isused to construct a standard curve. The amount of target sequenceproduct in the experimental wells is then compared to the amount of ICCproduct produced. By comparing the amount of product produced from thetarget sequence with that of the ICC standards, the startingconcentration of target sequences is determined.

EXAMPLE 6

[0091] In this Example, a PCR reaction including a known quantity of theICC is run as described in Examples 1 and 2. A variety of methods areknown by one skilled in the art for the quantification of plasmid DNA aswell as PCR production quantification (e.g., absorbance at 260 nm). Theamount of target PCR product can be quantified by comparison to theamount of ICC PCR product produced from a known amount of ICC added tothe target amplification reaction.

[0092] The internal control plasmid may also be used to performcalibration of inhibitory factors found in patient samples.

[0093] An Internal Control for Branched Oligonucleotide SignalAmplification

[0094] An embodiment of the present invention consists of a controlsequence wherein the entire internal sequence being reversed. In thisconfiguration, the present invention may be used as an internal controlfor a branched oligonucleotide signal amplification assay. The branchedoligonucleotide signal amplification assay uses a series of probes tobind and amplify a target sequence. For example, human immunodeficiencyvirus type (HIV-1) RNA was detected and quantified using a branched DNAsignal amplification by Pachl et al. (See Pachl et al., “Rapid andPrecise Quantification of HIV-1 RNA in Plasma Using a Branched DNASignal Amplification Assay,” Journal of Acquired Immune DeficiencySyndromes and Human Retrovirology, 8: 446-454 (1995); hereinincorporated by reference).

[0095] In the branched DNA assay (bDNA), the target is bound to a wellof a microtiter plate by oligonucleotide probes that bind to and capturethe target sequence. After binding, the bound target-capture probecomplexes are exposed to another target-capture probe that is alsocapable of binding a branched DNA molecule as well as the targetmolecule. An enzyme labeled branched DNA probe is then added to themixture and exposed to the substrate of the enzyme label. The signalobtained from this enzymatic reaction can be used to quantify the amountof target originally captured by the assay.

[0096] The present invention provides a method to control for internalinhibition of the bDNA assay. By using an internal control composed ofan inverted sequence of the target molecule, inhibition of primerbinding can be controlled. In one embodiment, an inverted controlplasmid is constructed and the RNA produced from this plasmid isintroduced along with the target sample into the microtitre well. Unlikein the previous embodiments, the same capture probes would not be usedfor both the control and the target sequences. However, due to thesimilarity of the sequences, the make up and binding of the controlprobes to the control sequences would accurately reflect the bindingthat occurs on the target molecule.

[0097] In a manner similar to the PCR example discussed above, thepresence of an internal control of inhibition would permit aninvestigator to more accurately arrive at result indicating a negativeor positive result.

EXAMPLE 7

[0098] Blood is obtained by routine phlebotomy techniques and tested forthe presence of HIV-1 RNA using a branched DNA signal amplificationassay. Heparin containing blood collection tubes should not be used asthe presence of heparin appears to negatively effect the concentrationHIV-1 RNA in the plasma. (See Pachl et al.). Plasma is obtained from theblood samples using centrifugation at 800 g for 10 minutes. The plasmais stored at less that −70° C.

[0099] Plasma specimens are treated by the addition of 50 :l of a 0.1%red polystyrene 0.5 :m bead suspension (Bangs Laboratories, Carmel, Id.)in 10 mM Tris-HCl pH 8.0, 1 mM EDTA to each plasma containing tube. Thesamples are centrifuged for 1 hour at 23,500 g at 2-8° C. Thesupernatant is then removed and the viral pellets are extracted for usein the branched DNA assay.

[0100] The virus pellets are extracted using 220 :l of Specimen WorkingReagent [400 mM LiCl, 100 mM HEPES pH 7.5, 8 mM EDTA, 1% lithium laurylsulfate, 12 :g/ml sonicated salmon sperm DNA, 0.04% Na azide, 0.04%Proclin 200 (Supelco, Bellefonte, Pa., U.S.A.), 2.2 mg/ml proteinase K,0.375 pmole/ml of each HIV-1 target probe or control probe to mediatecapture, 1.25 pmole/ml of each HIV-1 or control probe to bindamplifier]. The ICC control plasmid may be added to the sample viruspellet before extract or after the extraction procedure is completed.The ICC and the control primers may be kept separated from the targetsample and used in a separate well of the assay plate discussed below.

[0101] The mixture of virus pellet is then vortexed, incubated at 53° C.for 20 minutes to extract the viral RNA, vortexed again, and centrifugedat 23,500 g for 15 minutes to clarify the pellet extract. The clarifiedextract (200 μl) is then added to the assay wells of a 96-well assayplate which is coated with either HIV-1 capture probes or control probesor a mixture of the two. Other standards in addition to the ICCinhibition control may also be used.

[0102] The assay plate is then incubated at 53° C. to permit the bindingof the target or ICC control molecules to their respective probes. Thewells are then allowed to cool to room temperature for 10 minutes andare then washed twice with Wash A, a standard saline citrate (SSC)-0.1%SDS buffer. An amplification buffer containing 2.0 pmole/ml bDNAamplifier in Amplifier Diluent (50% horse serum, 1.3% SDS, 6mM Tris-HClpH 8.0, 5×SSC), is added along with 0.5 mg/ml proteinase K and isincubated at 65° C. for 2 hours. This incubation is followed by theaddition of 1 mM phenylmethylsulfonyl fluoride (PMSF) to inactivate theproteinase K, and 0.05% each Na azide and Proclin 300.

[0103] The wells are then sealed and incubated at 53° C. for 30 minutesin order to hybridize the bDNA amplifier molecules to the target-probeor control-probe complexes on the microwell surface. The wells weresubsequently cooled and washed as above, followed by the addition of 50:l of HIV Label Working Reagent (4 pmole/ml alkaline phosphatase-labeledprobe in Amplifier Diluent). The wells are then sealed and incubated at53° C. for 15 minutes to hybridize the alkaline phosphatase probe to theimmobilized bDNA amplifier molecules. The wells are then cooled as aboveand washed twice with Wash A followed by three washes with Wash B(0.1×SSC). A 50 :l volume of chemiluminescent substrate, an enzymetriggerable dioxetane substrate for alkaline phosphate (Lumiphos 530,Lumigen, Detroit, Mich., U.S.A.) is added to each well and incubated at37° C. for 30 minutes. Light emission is then measured in a luminometer.

[0104] An Internal Control for RNA Amplification Rreactions (RT-PCR)

[0105] In another embodiment, the positive control is a RNA molecule.When an RNA molecule is the target of a signal amplification assay, aRNA molecule should be used as a positive inhibition control. RT-PCRrefers to the use of reverse transcriptase in the PCR reaction. SinceRNA molecules are more labile to degradation than double stranded DNA,it is appropriate to control for degradation using the same type ofnucleic acid. In a preferred embodiment, a PCR based RNA amplificationassay is used.

EXAMPLE 8

[0106] The following protocol is based on Current Protocols in MoleculeBiology Volume 2, Chapter 15.4 (1995), which is incorporated herein byreference.

[0107] A target RNA molecule is used as a template in the PCR reaction.A sample of RNA is obtained through standard methods known in the art.The RNA used in the signal amplification reaction may be poly(A)+ RNA,or total RNA may be used. Alternatively cytoplasmic RNA may be used. Thetype of RNA used in the reaction will determine the type of control RNAused. The control RNA may also be added directly to the samplecontaining the target RNA for testing purposes.

[0108] A solution is prepared of 2 :g of RNA, 25 ng (3 pmol) cDNAprimer, and sufficient H₂O to bring the volume to 90 :l. After mixing,10 :l of 3 M sodium acetate, pH 5.5 and 200 :l of 100% are added. Thissolution is mixed and allowed to precipitate overnight at −20° C. or for15 minutes at −70° C. The sample is then centrifuged for 15 minutes athigh speed at 4° C. The resulting pellet is saved and the supernatant isdiscarded. The pellet is washed with 70% ethanol and centrifuged for 5minutes at high speed, room temperature. The supernatant is discarded.The pellet is dried briefly in a desiccator.

[0109] The following ingredients are added to the RNA pellet: 12 :l H₂O,4 :l 400 mM Tris-HCl, pH 8.3, and 4 :l 400 mM KCl. The solution isheated to 90° C. and then cooled slowly to 67° C. The sample is brieflymicrofuged to collect any condensate that may have formed and incubatedfor 3 hours at 52° C. Again, the sample is briefly microfuged to collectany condensate.

[0110] A complementary or cDNA molecule is synthesized in the next step.Twenty-nine microliters of reverse transcriptase buffer (50 mM Tris-HCl,pH 8.2, 5 mM MgCl2, 5 mM DTT, 50 mM KCl, 50 :g/ml BSA) and 0.5 :l (16 U)avian myeloblastosis virus (AMV) reverse transcriptase are combined.These reagents are mixed and incubated for 1 hour at 42° C. One hundredfifty microliters of 10 mM Tris-HCl/10 mM EDTA, pH 7.5 are combined andthe solution is mixed again. The solution is phenol extracted with 200:l buffered phenol and vortexed. The sample is microcentrifuged for 5minutes at high speed, and the aqueous phase is saved. The mixture ischloroform extracted with a solution of 24 :l chloroform:isoamyl alcoholand vortexed. The sample is microcentrifuged for 5 minutes at highspeed, and the aqueous phase is retained. The solution is precipitatedwith 20 :l of 2M sodium acetate, pH 5.5, and 500 :l of 100% ethanolovernight at −20° C. or for 15 minutes at −70° C. The sample ismicrofuged for 15 minutes at high speed for 4° C. and the supernatant isdiscarded. The pellet briefly dried and resuspended in 40 :l of H₂O.This material is the template in the PCR reaction.

[0111] Following precipitation of the cDNA, the target and controlmolecules are amplified using PCR. To a 5 :l sample of cDNA 5 :l of eachamplification primer (20:M each) is added. To that 4 :l of 5 mM dNTP mix(see PCR protocol above), 10 :l of 10×PCR amplification buffer and 70.5:l of H₂O is added and the reaction mixture is heated at 94° C. Thesample is microcentrifuged and 2.5 U of Taq DNA polymerase is added tothe reaction mixture. The solution is overlaid with 100 :l of mineraloil before the reaction is run. Forty or more cycles in an automatedthermocycler are performed to amplify the target molecules.

[0112] The reaction products are then assayed for the presence orabsence of control sequence as discussed in Example 3 or 5.

[0113] Enantiomeric and Reversed Amino Acid Sequence Sequences Used forInternal Controls

[0114] Another aspect of the present invention contemplates the use ofreversed amino acid sequences and enantiomeric sequence as controls forimmunological assays. Enantiomers are compounds that have the ability torotate the plane of plane-polarized light as it passes through asolution. Such compounds are asymmetric so that they can exist in twodifferent structural forms (D and L forms). Each of the structural formsexist as mirror images of each other and has the capability of rotatinglight in a particular direction. Proteins are naturally occurringpolymers of L-amino acids. Synthetic enantiomers of naturally occurringproteins and smaller peptide sequences can be readily chemicallysynthesized using D-forms of amino acids. These peptides can then beused as controls for immunoassays.

[0115] An important reagent in many immunoassays are antibodies.Monoclonal or polyclonal antibodies are raised to a specific amino acidsequence within the protein of interest. The techniques to raiseantibodies are well known in the art. For example, see Antibodies: ALaboratory Manual, (Harlow and Lane, Eds.), Cold Spring HarborLaboratory (1988). The amino acid sequence of a target protein,consisting of naturally occurring L-amino acids, is determined usingstandard techniques known in the art. An enantiomeric sequence,identical to the native epitope, only using D-amino acids is synthesizedand used to raise antibodies.

[0116] Chemical methods of peptide synthesis are well known in the art.The use of tertiary-butyloxycarbonyl blocking groups (t-Boc chemistry)or fluoromethoxy carbonyl blocking groups (Fmoc) are two suchmethodologies. Once individual D-amino acids are synthesized and fittedwith blocking groups, a short D-peptide is synthesized. In oneembodiment solid-phase synthesis is used. Following decoupling from thesynthetic resin the D-peptides are purified using high performanceliquid chromatography (HPLC). The conditions for such purificationdepend on the amino acids used to form the D-peptides. The peptidesequences are confirmed through amino acid sequencing techniques thatare also well known in the art.

[0117] Following the synthesis of the D-peptides, an antibody would beraised against it. Monoclonal antibody production is well known in theart. Briefly, a target animal, preferably a mouse, would be immunizedwith the D-peptides. After an appropriate number of boosterimmunizations, the spleen of the immunized animal is harvested and usedto create hybridomas using techniques well known in the field. Followinghybridoma generation, individual colonies are screened for antibodyproduction. Those colonies that produce active antibody can be expandedto produce large quantities of the monoclonal antibody. (See Antibodies,A Laboratory Manual, Chapter 7, eds. Ed Harlow & David Lane, Cold SpringHarbor Laboratory (1988), herein incorporated by reference).

[0118] In a preferred embodiment, the D-peptides and the monoclonalantibodies generated against them are used as a control for inhibition.Control D-peptides that are enantiomers to a known target antigen areadded to a sample in a detectable quantity. In a preferred embodimentthe control antigen is added in a range from 1 to 100 ng/well.Antibodies specific for both the target epitope and the controlD-peptides are added to the mixture. In a preferred embodiment the twoclasses of antibodies are bound to two different fluorescent beadpopulations so that the binding of control and target antibodies may bedetermined. As with the other embodiments of this invention, the absenceof binding of the control antibody to the control D-peptides wouldindicate inhibition of the assay.

[0119] Immunoassays are well known in the art and involve the detectionan antibody or an antigen. For example, a well-known form of immunoassayis the enzyme linked immunosorbent assay or ELISA assay. In thisimmunoassay an antigen is bound to the bottom of an assay plate andexposed to a sample of plasma that contains antibodies. The assay plateis then washed and then exposed to an anti-antibody antibody coupled tosome signal-producing molecule, like an enzyme. The assay plate is thenscreened for the presence of a signal. The presence of a signalindicates antibody binding and therefore the presence of antigen. Often,the level of signal produced can be used to determine the quantity ofantigen bound to the bottom of the well.

[0120] Assays such as the ELISA are extremely useful for screeningsamples for the presence of particular proteins. For example, if aperson has been exposed to HIV then it is likely that that person willcarry antibodies specific for certain HIV proteins. If that personwishes to donate blood, then their blood may be screened for antibodiesagainst HIV that would suggest that the donated blood was contaminatedwith HIV. On the other hand, a negative result could be improperlyinterpreted as the absence of HIV exposure if that negative result wasdue to inhibition of the assay. If some molecule was inhibiting theassay mechanism, an investigator could incorrectly conclude that thesample tested was free of HIV antibodies. An internal control forinhibition would permit investigators who monitor the blood supply toaccurately differentiate a true positive from a false positive testresult.

EXAMPLE 9

[0121] A blood sample is taken using standard techniques known in theart and tested for the presence of the HIV protein, gp24 protein.Antibodies to an epitope of the gp24 protein as well as its enantiomerare generated as discussed above. The blood sample is processed for usein an ELISA assay.

[0122] An ELISA assay is performed to test for the presence or absenceof the gp24 protein according to protocols well known in the art. Astandard microtiter plate containing positive and negative controls aswell as experimental wells is assembled. To control for inhibition, adetectable amount of gp24 enantiomer epitope is included in theexperimental wells of the assay. In one experimental well, an antibodyspecific for the gp24 epitope is used in the assay. In anotherexperimental well, an antibody specific for the gp24 enantiomer epitopeis used. Reagents that detect the presence of anti-gp24 or gp24enantiomer antibodies are then added to the assay. The results from theassay are then analyzed.

[0123] The presence of a negative response from the anti-gp24 containingwells of the assay suggests that the tested blood lacks detectable gp24.Nevertheless, the lack of signal from this well may instead result frominhibition of binding by the anti-gp24 antibody. To determine whetherinhibition is present in the experimental well, the results from theexperimental well assayed with the anti-gp24 enantiomer antibody areexamined. If a positive signal is present in this well, then there wasno inhibition, since the anti-gp24 enantiomer antibody reacted with itstarget epitope. On the other hand, if there is no signal from theanti-gp24 enantiomer antibody containing wells, then inhibition of theELISA assay may be present and the investigator may not conclude thatthe assayed blood sample is free from gp24.

[0124] An Internal Control for Nucleic Acid Amplification BasedAmplification (NASBA) and Transcription Mediated Amplification (TMA)

[0125] In another aspect of the present invention, the ICC may be usedas an internal inhibition control for a NASBA target amplificationassay. The NASBA and TMA technologies are isothermal nucleic acidamplification assays that are virtually identical in principle andpractice. These isothermal nucleic acid amplification assays useoligonucleotide probes, an RNA dependent polymerase and a reversetranscriptase to amplify a target sequence. An isothermal nucleic acidamplification assay, unlike PCR, is an isothermal amplification method,thus it does not cycle between high and low temperatures to facilitatetarget amplification. For example, the amplification of humanimmunodeficiency virus type (HIV-1) RNA was described using a NASBAsignal amplification. (See Kievits et al., “NASBA TMisothermal enzymaticin vitro nucleic acid amplification optimized for the diagnosis of HIV-1infection,” Journal of Virological Methods, 35: 273-286 (1991); hereinincorporated by reference).

[0126] In a NASBA assay, for example, the target RNA molecule is placedinto solution with oligonucleotide primers and two polymerases. Thetemperature of the sample is raised to prepare the target molecules foramplification. The temperature is then lowered to permit the primer bindto the template. After binding the target, a DNA molecule complementaryto the target sequence is synthesized using a reverse transcriptase,such as the AMV-reverse transcriptase. Subsequent to this synthesis, theRNA-DNA hybrid molecule is digested with RNase H, removing the RNAstrand. A complementary strand to the single stranded DNA targetsequence is synthesized through another round of DNA polymerizationusing a reverse transcriptase enzyme.

[0127] Following the creation of this double stranded DNA molecule,single stranded RNA is synthesized from the double stranded DNA templatewith T7 RNA polymerase. This step amplifies the original target sequence100 to 1000-fold. Simultaneously, new double stranded DNA templates arebeing synthesized from the replicated single stranded RNA templatesproduced in the first round of amplification. From this series ofreactions, the target sequence is amplified. The products of thisreaction can be assayed to determine the presence or absence of a targetmolecule. Quantitation of the product created by the reaction ispossible using the methods described herein. Also, with the inclusion ofthe ICC sequence of the present invention, a clinician using NASBA toassay for the presence or absence of a target molecule could accuratelydetermine if inhibition of the reaction occurred, resulting in apossibly false negative response.

EXAMPLE 10

[0128] Blood is obtained by routine phlebotomy techniques and tested forthe presence of HIV-1 RNA using a NASBA signal amplification assay. Anucleic acid sample is isolated from the plasma using generally knownmethods. (See Boom et al., “A rapid and simple method for purificationof nucleic acids,” J. Clin. Microbiol. 28, 495-503 (1990); hereinincorporated by reference). The isolated nucleic acid is resuspended inwater and stored at −70° C.

[0129] Two microliters of isolated nucleic acid solution are mixed with23 :l of a reaction mixture containing (at a final concentration in a 25ul reaction mixture): 40 mM Tris, pH 8.5, 12 mM MgCl₂, 42 mM KCl, 15%v/v DMSO, 1 mM each of dNTP, 2 mM each NTP, 0.2 :M Primer 1, 0.2 :MPrimer 2, 2 :l of ICC internal control RNA in a known quantity at asufficiently high concentration to provide a signal from the NASBAassay.

[0130] The sample is incubated at 65° C. for 5 minutes, destabilizingany secondary structures in the nucleic acid target. The mixture iscooled to 41° C. and the primers are thus annealed to the template. Theamplification reaction is started by adding 2 :l enzyme mixture (0.1:g/:l BSA, 0.1 units RNase H, 40 units T7 RNA polymerase and 8 unitsAMV-reverse transcriptase). The reaction mixture is incubated at 41° C.for 90 minutes. The reaction mixture is then assayed for target andcontrol signal amplification. The absence of a signal from the ICCcontrol signal indicates inhibition of the signal amplificationreaction.

EXAMPLE 11

[0131] Transcription-Mediated Amplification (TMA) Assay

[0132] A blood sample is tested for the presence of the HSV gB genetranscript according to the TMA protocol described in Stary, A., et al.,“Performance of transcription-mediated amplification and ligase chainreaction assays for detection of chlamydial infection in urogenitalsamples obtained by invasive and noninvasive methods.” J Clin Microbiol.36(9):2666-70 (1998). A negative response from an experimental samplecontaining the ICC control indicates the presence of inhibtion.

[0133] Molecular Beacons

[0134] The term molecular beacons relates to an assay system thatutilizes probes that fluoresce upon hybridization with a targetsequence. Molecular beacons are discussed in Tyagi & Kramer, “MolecularBeacons: Probes that Fluoresce upon Hybridization,” NatureBiotechnology, 14:303-308 (1996) and in Giesenfor, et al., “Molecularbeacons: a new approach for semiautomated mutation analysis,” ClinicalChemistry 44:482-486 (1998). The principle of this assay system involvesa probe that consists of a stem-and-loop structure. The loop portion ofthe molecule is a probe sequence complementary to a predetermined targetsequence. The stem is formed by annealing on either side of the probesequence two complementary arm sequences that are unrelated to thetarget sequence. A fluorophore is attached to the end of one arm and aquenching moiety is attached to the end of the other arm. The stem keepsthese two moieties in close proximity to each other and quenches thesignal from the fluorophore. Once the molecular beacon binds to itstarget, the fluorophore is separated from the quencher, permitting asignal to be generated from the probe. Thus, the probe undergoes aspontaneous conformational change that forces the arm sequences apart,thereby moving the fluorophore and the quencher away from each other andresulting in the generation of fluorescence.

[0135] As with any assay system, the production of a signal provides abasis to conclude that the target sequence is present in theexperimental sample. The converse does not necessarily hold, however,since the absence of a signal in a hybridization assay may result fromamplification inhibition rather than the absence of target from theassayed sample. The present invention may be used as an internalinhibition control to determine whether the absence of a signal is alegitimate negative result or merely a result of inhibition.

EXAMPLE 12

[0136] A blood sample is prepared from an individual to be screened forthe presence of HSV gB. The blood sample is obtained and prepared bymethods well known in the art. Molecular probes specific for the HSV gB(SEQ. I.D. NO. 7) and ICC (SEQ. I.D. NO. 8) sequences are constructedaccording to the method of Tyagi and Kramer.

[0137] One hundred and fifty μl of a 170 nM solution of molecular beaconprobe SEQ ID NO 7 and 8 are separately dissolved in 100 mM Tris-HCl (pH8) containing 1 mM MgCl₂ that is maintained at 25° C. The fluorescenceof each probe solution is monitored at 490 nm with time in an LS-5Bspectrofluorometer (Perkin Elmer), using 1 cm path length QS curvettes(Hellma) whose temperature is controlled by a circulating water bath.There is no change in fluorescence with time, so a sample containing5-fold molar excess of target sequence and ICC are added to curvettescontaining the probes. The level of fluorescence emitted is recorded.

[0138] The temperature of the sample is gradually increased to denaturethe probe and promote hybridization from 25° C. to 75° C. at a rate of2° C./minute. As the temperature increases, the amount of fluorescencedetected from the samples containing the ICC and ICC specific probeincreases. In the experimental reaction mix, no signal is detected fromthe HSV gB probe. Since there is signal from the ICC probe, there is noinhibition of this experimental preparation.

EXAMPLE 13

[0139] Molecular Beacons and Real-Time PCR Analysis

[0140] A blood sample is prepared from an individual to be screened forthe presence of HSV gB using real-time PCR. This procedure entailsmonitoring the generation of fluorescence during the various PCR cyclesusing an ABI 7700 Sequence Detector (Perkin-Elmer/Applied BioSystems).The blood sample is obtained and prepared for use in PCR by methods wellknown in the art. Molecular beacons specific for the HSV gB (SEQ. I.D.NO. 7) and ICC (SEQ. I.D. NO. 8) sequences are constructed according tothe method of Tyagi and Kramer. The molecular beacons are labeled withdifferent fluorescent probes to emit different signals when bound.Alternatively, the beacons may be labeled with the same probe and toadded to different reaction mixtures to monitor target sequencesynthesis and control sequence synthesis separately.

[0141] The PCR samples are assembled as discussed in the Examples above.PCR primers, SEQ ID NOS 3 and 4 are used to amplify the target andcontrol sequences. The PCR reaction buffer is as described above withthe addition of a fluorescent dye at 60 nmol final concentration (ROX,Perkin-Elmer). The molecular beacons are added directly to the PCR mix.To the individual PCR reactions are added blood samples or controls. Toeach of these is added the PCR mix as well as a sample of ICC. The PCRtubes containing the individual reactions are then subjected tothermocycling.

[0142] At 95° C., the molecular beacons are denatured and have a randomcoil structure, allowing full fluorescence. During the decrease of thetemperature in the PCR cycle, the formation of hairpins occurs, whichcauses a drop in fluorescence. In samples containing lacking the HSVtarget sequence the molecular beacons fails to bind to theircomplementary sequences and there is no increase in the fluorescencedetected. However, in those experimental reactions containing themolecular beacon specific for the control sequence, molecular beaconbinding to their complementary sequences does occur and fluorescenceincreases.

[0143] From these results the investigator may reasonably conclude thatthere is no signal inhibition and the negative result observed reflectsan absence of target sequence in the experimental PCR reaction.

We claim:
 1. An internal control cassette for use in a polynucleotidedetection assay in which a target sequence is detected, said targetsequence having primer binding sites flanking an internal targetsequence, said cassette comprising primer binding sites flanking aninternal control sequence, wherein the internal control sequencecomprises said internal target sequence in a reversed orientation.